Characterization of the Tetrahymena thermophila 22S dynein heavy chains

Tracie Moniece Gibson, Purdue University

Abstract

Dynein is a large multiprotein complex that contains several subunits. At least 12 axonemal dynein isoforms participate in the bending of cilia and eukaryotic flagella. There are two classes of axonernal dynein: inner and outer arms. Outer arm dynein from Tetrahymena thermophila is composed of three different heavy chains, termed α, β, and γ. Recent studies suggest that the heavy chains play distinct roles in ciliary bending. These studies describe the biochemical and genetic approaches that were used to isolate and characterize Tetrahymena 22S dynein heavy chains. In order to decipher the contribution of individual heavy chains to the motor activity of dynein, it is necessary to separate the complex. We developed a novel approach to partially dissociate the heavy chains and performed functional analysis of the dissociated complexes. Controlled dialysis of 22S dynein into 4 M urea followed by centrifugation through a 5–25% sucrose gradient resulted in the dissociation of the complex into two functional dynein species: the 14S species contained the cc heavy chain, and the 19S dynein species contained the β/γ dimer. Functional studies revealed that the heavy chains retained MgATPase activity. The 22S, 19S, and 14S dynein species bound microtubules, but the 14S dynein species did not demonstrate ATP-sensitive binding. Finally, in vitro motility assays revealed that the β/γ translocated microtubules at a similar rate to intact 22S dynein. However, the isolated a heavy chain did not translocate microtubules. A genetic approach was used to understand the contribution of the β heavy chain to the motor activity of dynein. Macronuclear gene disruption was achieved by biolistic bombardment and transformants were selected in the neomycin analogue, paromomycin. The KO-β transformants were almost immotile. Biochemical studies of isolated ciliary high salt extracts from KO-β revealed that all three heavy chains of outer arm dynein were depleted. Our results are consistent with the model that the α heavy chain mediates rigor and not motility. In contrast the β heavy chain plays an important role in the motor activity of outer arm dynein.

Degree

Ph.D.

Advisors

Asai, Purdue University.

Subject Area

Cellular biology|Molecular biology

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